The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a signal peptide followed by a Pexel/HT erythrocyte trafficking motif. This catalog includes two large families of proteins that are the subject of this proposal: the predicted 2 transmembrane (2TM) proteins that likely function within the erythrocyte membrane, including the RIFIN, STEVOR and Pfmc-2TM families, and the PHIST domain family which likely function within the erythrocyte cytoplasm. The human malaria parasite, P. vivax, and the rodent malaria parasites possess large families of proteins which are topologically similar to the 2TM proteins and also harbor Pexel/HT motifs, suggesting that the function of the 2TM proteins is widely conserved in Plasmodium. We have shown using primary and second generation clonal lines of the P. falciparum isolate, NF54 that expression of stevor and Pfmc-2TM families is clonally variant and undergoes switching. Moreover, the STEVOR and Pfmc-2TM families possess a loop between the 2TM domains that is hypervariable both between paralogs and across isolate boundaries, suggesting that it is exposed to host immune pressure at the erythrocyte surface. Thus host immune pressure has likely driven the amplification and antigenic diversification of the 2TM protein families, and a mechanism of gene expression switching insures that immune responses do not eliminate parasite infections. The topological and sequence similarity of the 2TM proteins to subunits of ion channels prompted us to consider that they might encode the new permeability pathways (NPPs) of infected erythrocytes. We propose here to test this hypothesis using genetically manipulated parasite lines that either dominantly express epitope-tagged versions of 2TM proteins or are globally knocked down in 2TM protein expression via conscription of specific transcription factors. These parasite lines will be used phenotypic assays, such as sorbitol lysis and whole-cell patch clamp. We also present evidence that the methodology of gene knockdown via transcription factor conscription can be used to address the function of other sub-telomeric gene families in P. falciparum, and thereby are likely to contribute to our understanding of parasite-encoded modifications of the infected erythrocyte. We present preliminary studies on the gene expression and cellular localization of select PHIST domain proteins and propose to pursue their function via targeted gene disruption as well as determination of predicted protein-protein interactions within the erythrocyte cytoplasm. In this manner we hope to generate hypotheses on the identity of the functional pressures which have driven the amplification of the PHIST domain family to over 6 members in P. falciparum. PUBLIC HEALTH RELEVANCE: Malaria remains a public health concern to approximately 40% of the world's population, particularly in the face of increasing drug resistance and the continued absence of an effective vaccine. This proposal focuses on two amplified sub-telomeric gene families, the first encoding the 2TM proteins which uniformly possess signal peptides, Pexel/HT erythrocyte trafficking motifs, and 2 transmembrane (2TM) regions that indicate an integral membrane topology. The second gene family encodes the PHIST domain proteins which include over 60 members and are predicted to function with the erythrocyte cytoplasm. The 2TM proteins are conserved in all species of Plasmodium, not as orthologous families but rather as topologically conserved integral membrane proteins. Thus their function, likely at the erythrocyte surface, is predicted to all be conserved across the Plasmodium clade. In this proposal we argue that the profound amplification of the 2TM gene families, their diversity between paralogs and across isolate boundaries, localization to the erythrocyte membrane, and topological similarity to sub-units of ion channels, combine to make them premier candidates for comprising the new permeability pathways in infected erythrocytes. We describe a spectrum of studies in which to test this hypothesis, including new methods to knockdown expression of gene families. The expression, cellular localization, and phenotype following targeted gene disruption will be studied for select PHIST domain proteins toward an understanding of their function within the erythrocyte cytoplasm.